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					                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin




                                     IKDT Laboratory
       Your Partner for Molecular Diagnostics in
        Primary and Secondary Cardiomyopathies


IKDT Manual 2009
Content

1. General

2. Performance

3. Quality Assurance - Quality Management

4. IKDT Staff

5. Pre-analytical treatment of patient samples

6. Histology

7. Immunohistochemistry

8. Molecular Diagnostics
    • Detection of viral genomes by nested-PCR
    • Sequencing of viral gene fragments
    • Quantification of viral load

9. Autoimmunity Testing

10. Immunoassays

Summary

  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


1. General
In order to define the current role of endomyocardial biopsy (EMB) in the management of
cardiovascular disease, a multidisciplinary group of experts in cardiomyopathies and cardiovascular
pathology were convened by the American Heart Association (AHA), the American College of
Cardiology (ACC), and the European Society of Cardiology (ESC). In form of 14 clinical scenarios
were published recommandations for taking EMBs and the requested examinations in October
2007 (Circulation 2007;116;2216-2233).
The IKDT Institut Kardiale Diagnostik und Therapie GmbH is performing routine and specialized
diagnostics of cardiologic diseases in close cooperation with Department of Cardiology and
Pulmonology, Medical Clinic II in Berlin. IKDT is performing nearly all requested examinations on
EMBs following the international recommandations.

It was founded in August 2002 and started its laboratory work in January 2003. IKDT lab is
performing diagnostic examination of endomyocardial biopsies. Its service is offered to all
cardiological hospital departments in Germany and Europe.
Today IKDT is one of the leading laboratories on viral infections of heart muscle tissue. Molecular
diagnostics of patient samples and also research samples is ensured by sufficient and well-educated
and highly motivated technicians. All necessary analytical devices are existing in IKDT lab. New
diagnostic tests will be adapted from currently applied methods.
IKDT lab is the only clinical laboratory in Germany which is accredited by College of American
Pathologists (CAP) to perform extended diagnostics of endomyocardial biopsies. CAP is the only
organization which is approved by US Food and Drug Administration (FDA) for accreditation of
diagnostic labs outside of the US. The Laboratory Accreditation Program (LAP) includes a regular,
biannual peer-review of laboratory and intermediate self-inspection by laboratory director.
Implemented QM systems is following the GLP/GCP guidelines and Clinical Laboratory
Improvement Amendments of 1988 (CLIA-88).
From 2003 to 2006, IKDT was the core lab for molecular diagnostics of endomyocardial biopsies
during the European trial on treatment of chronic cardiomyopathies by Betainterferon, which was
organized by Department of Cardiology and Pulmonology, Medical Clinic II of Charite University
Hospital Benjamin Franklin (CBF) in collaboration with Schering AG Berlin.

2. Performance
IKDT is performing diagnostics on endomyocardial biopsies on requests of hospital-affiliated
institutions or private doctor offices. For routine diagnostics will be covered three main topics: 1.
Histology, 2. Immunohistochemistry and 3. Molecular Virology. Four endomyocardial biopsies at
minimum are required for whole routine procedure. Increased number (6-8) will be benefical for
diagnostic accuracy. Sampling error is not negligible for detection of cardiotropic viruses.
In the currently finalized European clinical trial on Beta-Interferon treatment of chronic
cardiomyopathy (BICC) were included about 400 patients, 9 myocardial biopsies from each patient,
which have to be analyzed by three different methods: PCR for viral genomes, surgical histology
and immuno-histochemistry.

  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


The histological/immunohistochemical examination is performed within 2 working days, whereas
routine diagnostics of EMBs is performed completely within 5 days.

Additionally IKDT is offering testing on circulating antibodies to heart proteins in patient. For
these assays, based on reactivity of patient antibodies against myocardial structure components
like nucleus, basal membran, mitochondria, striated muscles, are requested fresh serum or plasma.

3. Quality Assurance - Quality Management
The institute disposes over a management with a rigid organization which does not allow any
undefined deviations. The laboratory director of IKDT and representatives of consulting CBF board
are responsible for the assurance of the quality of examination results. The QM system is covering
internationally accepted GLP/GCP CLIA-88 guidelines.

From the beginning in-house established QM system was focused on Laboratory Accreditation
Program of College of American Pathologists (CAP). Their Laboratory Accreditation Program
(LAP) was established in 1961. In 1995 CAP received approval as an accrediting organization
under the Clinical Laboratory Improvement Amendments of 1988 by the Centers for Medicare and
Medicaid Services (CMS), an agency within the U.S. Department of Health and Human Services. In
2001, this approval was extended for an additional six years, through September 2007.




Figure 1: Certificate of IKDT lab accreditation by College of American Pathologists (CAP)

Since 2003 IKDT lab is accredited by CAP to perform endomyocardial biopsy diagnostics under
certified conditions. CAP accreditation is the only certification process outside of USA which is
accepted by US Food and Drug administration (FDA).



  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


The accreditation programs examine pre-analytical, analytical and post-analytical aspects of quality
management (QM) in the laboratory. This includes the performance and monitoring of general
quality control (QC), test methodologies and specifications, reagents, controls and media,
equipment, specimen handling, test reporting and internal performance assessment, and external
proficiency testing. In addition, personnel requirements, safety, document management and other
management practices are included in the inspection process. Laboratories that meet accreditation
requirements distinguish themselves as quality laboratories (Fig.1).

The objective is efficient sample processing in a short time respecting a high quality standard of
data acquisition by latest state of the art modern diagnostic methods and following supplement of
submitting institution with detailed data report.
IKDT disposes over sufficient and suitable rooms as well as modern facilities with new equipment
and devices which are regularly checked for proper functioning based on servicing contracts. A
sufficient number of employees is available to carry out the necessary work. Explicit standard
operating instructions (SOPs) are available for all equipment and procedure.
The personnel is qualified correspondingly to all areas and will also in future attend courses for
continued medical education (CME).




Figure 2: Certificates of successful participation at national INSTAND NAT survey

PCR results most critical for resulting treatment of patients in hospitals or by drug therapy. Main
focus is set on the permanent control of achieved PCR result. Detection of virus infection in
endomyocardial biopsies is a multi-step-procedure, whereas each step is essential for final result.

IKDT is participating three times per year at national surveys on nucleic acid amplification
techniques (NAT) for virus detection organized by INSTAND e.V, Düsseldorf. Through this
program, the INSTAND provides individual laboratories with unknown specimens for testing. The
participants analyze the specimens and return the results to the INSTAND for evaluation. In turn,
each participating laboratory receives a report of their performance and a certificate of successful
  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin
participation as well as a report summarizing the results of all participating laboratories. IKDT use
these survey for evaluation of test and its accuracy. IKDT passed the existing surveys for all
corresponding viruses successfully (Fig. 2).

4. The IKDT Staff

The managing and laboratory director Dr. Dirk Lassner is a biochemist with long time experience in
molecular biology and clinical chemistry. Prof. Dr. Ulrich Gross, former director and vice-director
of Institute of Pathology at CBF, is responsible as medical director for whole laboratory and for
histological examination of endomyocardial biopsies.
Beside medical and managing directors there are currently 3 medical technical laboratory assistants
and one secretary. The four physicians of the consulting board of the Department of Cardiology and
Pulmonology, Medical Clinic II of Charite University Hospital Benjamin Franklin (CBF) support
IKDT lab in validation of diagnostic findings, formulation of diagnostic reports and in clinical
recommendations for submitting physicians.

Scientific Consulting Board of CBF:
Prof. Dr. Heinz-Peter Schultheiss
Prof. Dr. Matthias Pauschinger
Dr. Uwe Kühl

5. Pre-analytical treatment of patient material
The IKDT performs diagnostic tests for detection of inflammatory processes or viral infection of
human myocard biopsies under stringent compliance of FDA guidelines. The diagnostic spectrum
includes histology, immunhistochemical analysis and PCR assays for detection of viral genomes.
Myocardial biopsies in native or fixed form are the preferred sample materials for these tests. For
suitable assays and results it is important that the submitted material undergoes a well defined pre-
analytic treatment (Tab.1).

For endomyocardial biopsies should be used a novel reagent for conservation by ambient
temperature. RNAlater is an aqueous, non-toxic tissue storage reagent that stabilizes and protects
cellular RNA in intact, unfrozen tissue samples. RNAlater eliminates the need to immediately
process tissue samples or to freeze samples in liquid nitrogen for later processing. RNAlater is also
suitable for preparation for genomic DNA, histological examination and immunohistochemistry.
Tissue pieces can be harvested and submerged in RNAlater for storage without jeopardizing the
quality or quantity of RNA obtained after subsequent RNA isolation. RNAlater can be added to cell
pellets and even cells in medium. The samples can then be stored frozen or unfrozen.
The endomyocardial biopsies (less than 0.5 cm in any one dimension) are simply submerged in
approximately 0.5 ml of RNAlater at room temperature. Please submerge biopsies by inverting tube
5 times. The solution permeates the cells, stabilizing the RNA. Preferably, samples should be sent
directly at ambient temperature to IKDT lab or stored at +4°C before transport.. The transport
could be performed in a padded envelope by conventional mail. After reception in IKDT lab, the
sample will be stored at +4°C for one night and then can be stored at 4°C for up to a month or at
25°C for up to a week or at -20 to -80°C indefinitely (the tissue does not freeze).
  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Never freeze or treat the biopsies by other fixatives before RNAlater fixation!

RNAlater is only suitable for very small tissue samples (biopsies). Larger piece (explanted heart
samples) has to be divided rapidly in small aliquots or should be cryo-conserved in liquid nitrogen.


Table 1: Proposed pre-analytical treatment of endomyocardial biopsies for diagnostics in IKDT lab
Submitted               Pre-analytic                  Detection                     Native/ fixed                 Shipment
material                treatment                     method
Myocardial              RNAlater                      PCR & Histology &             Fixed in RNAlater             Ambient temp
biopsies                                              Immunohistochemistry
Alternative
pre-treatment
Myocardial              frozen in liquid              PCR & Histology &             native                        on dry ice
biopsies                nitrogen                      Immunohistochemistry
Myocardial              fixed    in    4-5%             Only for histology !        fixed in                      Ambient temp,
biopsies                buffered formalin                                           formalin                      Do not freeze!

IKDT lab is providing submitting institutions by screw-cup tubes filled with RNAlater for
immediate use. Taken biopsies fixed in RNAlater should be transferred to IKDT for diagnostic
procedure.


Additional analysis

Detection of systemic viral infections or cytokine profiles is performed by analysis of peripheral
blood fractions. DNA or RNA from peripheral blood cells is examined by nested- and QPCR on
presence of viral genomes for exclusion or confirmation of systemic infection. EDTA-blood is
requested for detection of systemic viral infection (Tab. 2).
Predominating immune response in patient is evaluated by quantification of different sets of human
chemokines or cytokines in plasma or sera by ELISA tests. Plasma for immunological tests should
be collected with additives EDTA, aliquoted immediately in Eppendorf vials (0.2-0.5 ml per tube)
and stored till use at minimum at -20°C or preferable colder (-80°C). Serum for immunological tests
should be collected without any additives. Transfer to IKDT lab should be performed on dry ice.
Cytokines are very thermosensitive and will be destroyed after second thawing cycle (table 2).

Table 2: Proposed pre-analytical treatment of peripheral blood fractions for diagnostics in IKDT lab
Submitted               Pre-analytic                  Detection                     Native/ fixed                 Shipment
material                treatment                     method
Blood                   EDTA-tubes                    PCR                           native                        +4°C or ambient
Serum/Plasma            EDTA-tubes                    Immunology                    native                        frozen, on dry
                                                                                                                  ice below -20°C




  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


6. Histology
For histological examinations 4-5µm thick sections are prepared of paraffin-embedded biopsies by
cutting with the rotary microtome. For each staining procedure on one slide are placed between 3-8
serial sections. Routine diagnostics includes always HE, PAS and Elastica v. Weigert staining. The
staining of specimens, with the exception of special stainings, is always carried out in the staining
machines (Fig. 3).




                                                                                              Endokarditis
                   Endo- und Myokarditis nach Löffler       Aktive Myokarditis mit Nekrosen
                                                        A   B                                                                            C
Figure 3. Histological examination of Löffler’s endo and myocarditis (A), acute myocarditis with
          necrosis (B) and endocarditis (C)

Special staining for amyloid (Congo red), calcium (v. Kossa), acid mucosubstances (Alcian Blue)
and iron (Prussian Blue reaction) will be added in clinically suspected cases or on request of
submitting physician (Fig. 4).




      A                                                     B                                        C
Figure 4. Special stains of amyloid with Congo Red visualized in polarized light (A), von Kossa
          stain for calcium (B) and Alcian Blue for acid mucosubstances (C)

All histological examinations are performed by medical director Prof. Gross, which was over many
years Director of Institute of Pathology of CBF. Histological examination in IKDT lab follows
Dallas criteria for exclusion of acute or active myocarditis in examined biopsy sample. Main focus
is oriented on detection of myocytolysis in combination with leukocyctic infiltrates.
Observations are fixed as paperwork and as digitally printed colour-photographs which are saved
and stored on the data server as TIF-files or JPG-Files. Morphologic characteristics of stained
endomyocardial tissue (e.g. diameter of cardiomyocytes, size and quality of biopsy, fibrosis, fatty
tissue, capillaries) are rated by numeric scaling and the corresponding values are fixed on written
examination protocol and in the electronic IKDT database.
  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin



7. Immunohistochemistry
Immunohistological diagnostics are based on application of specific primary antibodies on cryo-
fixed tissue section and following detection of coupled primary antibody by secondary antibody.
Secondary antibody is conjugated with enzyme complex which could produce a precipitating
coloured complex after use of staining solution.
For immuno-histochemical examinations sections are prepared of cryo-embedded biopsies by use of
cryostat microtome. Therefore endomyocardial biopsy will be placed on pre-cooled (-20°C)
metallic tray and covered completely by plastination glue Tissue-Tek. Tissue-Tek is freezing down
immediately and preserve a hard consistence of embedded tissue.
Generally, cutting is performed for 3-5 slides for each antibody (about 20 cryo sections per patient)
before immuno-staining is started. Then separated areas are processed with different antibodies
accompanied with appropriate blocking and incubation steps and finally stained by an enzymatic
conversion of dye AEC for producing red-colored immunospots for following microscopic
examination. Second antibody and the colorimetric substrate are pre-mixed and well optimized for
following digital image analysis. The final counterstaining of cryo sections is always carried out in
staining machine (HE staining).
One microscopic slide is finally containing separated areas for 2 different antibodies. Hereby each
following layer of cryo sections is placed in the field for the next antibody detection, i.e. in any area
there are about 6 to 8 serial cryo sections. This cutting procedure ensure the more detailed analysis
by simultane staining of different levels of biopsy by various antibodies.

Four sets of immunohistochemical staining are offered for specialized diagnostics of heart muscle
tissue, whereas only the sets IC1-Heart muscle inflammation is proposed to perform in routine
diagnostics procedure. Set IC2-Activation marker/Viral proteins is recommended in clinically
suspected cases with high viral load of corresponding cardiotropic virus (Fig. 5).

IC1: Heart muscle inflammation (CD3, CD11a, CD11b, Perforin, HLA class 1, CD54)
IC2: Activation marker/Viral proteins (CD45R0, 27E10, CD69, CD106, EV-VP1, PVB19, HHV6)
IC3 - Remodeling of extracellular matrix (Collagen 1, 3, 4, CD29, MMP-9, TIMP-1)
IC4 - Cellular cytokine (IFN-gamma, TNF alpha, osteopontin, IL-10, IL-12, MCP-1)




   A                                                  B                                         C
Figure 5: Immunostaining of giant cell myocarditis with CD3 (A), detection of PVB19 positive
myocytes by VP2 antibody (B) and HHV6 infected cells (C) (see arrows)

  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Coloured immunospots are counted digitally by application of in-house established digital imaging
analysis software for calculating area fractions, numbers of immuno-spots and area of myocardial
tissue (routine diagnostics). Values for counting are fixed by inclusion of digitally produced values
in electronic database for in the final report. These report contains also numeric values for
morphological characteristics like biopsy size, quality, fibrosis etc.
Physician in IKDT controls the digital results by additional examination and prepares the report for
immunohistochemistry including laser print of colour photograph for the submitting physician.
In general, immunohistochemical staining is performed on frozen sections of a second biopsy, not
identical to histological examination. This procedure is benefical to reduce mis-interpretation by
evaluation of only one biopsy.


8. Molecular Diagnostics
The molecular diagnostic approach of EMBs are based on detection, quantification and sequencing
of viral genomes. With permantly increasing number of virus tests IKDT is focused on common
cardiotropic viruses which are described as responsible triggers of heart failure problems.
Established virus PCR detection methods of IKDT lab are listed in Table 3.

Test on cardiotropic viruses are based on qualitative detection of virus by nested-PCR and
quantification of virus load by quantitative TaqMan PCR. Depending on the 2 types of viral nucleic
acids we perform the isolation of DNA or RNA in separate extraction procedures.

Table 3: Established tests for cardiotropic viruses in IKDT lab
                                                 Nucleic   nested-        Subtyps / Sequencing of                         Determination of
Virus                                            Acid      PCR     TaqMan variants  positive PCR                          virus subtyp by
Parvovirus B19                                   DNA       X       X      G1, G2    yes                                   sequencing
Adenovirus                                       DNA       X       X      52        yes                                   sequencing
                                                 DNA,
Human Herpesvirus 6                              RNA       X        X            A and B          yes                     sequencing
Cytomegalovirus                                  DNA       X                     no               yes
Epstein-Barr-Virus                               DNA       X        X            no               yes
Herpes simplex virus 1 and 2                     DNA                X            1 and 2                                  TaqMan
Coxsackievirus                                   RNA       X        X            various          yes                     sequencing
Influenza                                        RNA                X            A and B                                  TaqMan
Measles                                          RNA                X            no

In order to calculate and standardize the estimation the virus load in small EMBs (viral genomes per
µg human ) IKDT lab apply the most accurate QUANTIFILER TaqMan test (Applied Biosystems,
USA), which was primary developed for forensics to detect minute traces of DNA.

All amplified virus genomes were sequenced for determination of existing virus subtype or
infectious variants. We apply double strand sequencing and subsequent manual alignment against
in-house reference files and international NCBI database (Fig. 8).

  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Isolation of DNA or RNA is performed from different emdomyocardial biopsies in a parallel
manner. During isolation procedure each staff member has to care for nuclease-free working
conditions (sterile tips, DEPC-treated water, often change of gloves etc.). After DNA extraction the
amount of isolated DNA has to be measured by special TaqMan assay. RNA is completely
transcribed into cDNA after DNAse digestion. Finally both nucleic acid fractions are existing as
DNA molecules and by this way better conserved from RNAse / DNase digestion.

Detection of viral genomes by nested-PCR

Polymerase chain reaction (PCR) is an artifical method for selective amplification of any desired
genome fragment in a very short time by use of a thermal cycler and thermostabile Taq DNA
polymerase. We apply this method for detection of Adenovirus (ADV)-, Coxsackievirus (CVB),
Epstein-Barr-Virus (EBV), ParvoB19-Virus (PVB) and human Herpesvirus 6 (HHV6) gene
sequences in endomyocardial biopsies.

                                                      Endomyocardium

                              Nucleic Acid Extraction (RNA or DNA)

                                            1. PCR (long fragment)


                                            nested-PCR (short fragment)


                                                 Analysis of PCR products
                                                  (Gel-Electrophoresis)


                       Sequencing                                                       TaqMan PCR
Figure 6: Flowchart of diagnostic procedure for detection of cardiotropic viruses

For all routineously analyzed cardiotropic viruses are used nested-PCR protocols consisting of two
sequentially performed PCR assays, where the amplicon of first assay is the template for second
reaction. This procedure is highly sensitive and enables us to detect very low copy numbers of viral
genes (ultrasensitive). As amplification control there are simultaneously processed serial dilutions
of a corresponding DNA-standard for checking PCR process.
Amplified PCR reactions are separated on agarose gel electrophoresis in ethidium bromide
containing buffer. This allows the subsequent visualisation of generated PCR product by UV
fluorescence. Amplicons with the same length size as the co-amplified standards are estimated as
positive signals and correspond to existing viral infection of myocardium (Fig. 7).
  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin




Figure 7: Gel electrophoresis of Coxsackievirus-nested-PCR (arrows – infected patients)

After nested-PCR aliquots of PCR samples are analysed by agarose gel electrophoresis. To the gel
is added ethidiumbromide for following staining in UV light (Fig. 7).
Documentation of PCR results is done by photographs made with digital camera in the gel
documentation system and a printout with a thermal printer. These printouts were ticked to
corresponding lab book for long-term documentation.

QPCR and sequencing data are generated by certified, electronic software. All raw data and export
files are stored on CD or streamer cassette for long-term storage following GLP/GCP guidelines.

Sequencing of viral genomes

All positive PCR reactions are sequenced as quality control of preceding nested-PCR and for
detection of amplified virus subtype. The generated sequences are checked by manual alignment
with PHYDE software (Institute of Botanics, Bonn/Dresden) and online with NCBI database for
confirmation of corresponding virus strain and / or estimation of specific virus subtypes or variants
(Fig. 8).


   A                                                                      B




Figure 8: Sequence fragment of Coxsackievirus (A) and alignment of PVB19 genotypes (B)




  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Quantification of viral load

Monitoring of successful treatment or therapy of infected patients has to be accompanied by
estimation of viral load in EMBs at different time points. Viral load is the ratio of viral genome

copies to associated amount of extracted myocardial tissue. In IKDT lab human genomic DNA , as
counterpart of extracted biopsy, was measured by quantitative TaqMan assay, which is
recommended for analysis of DNA amount in forensic traces by FDA.




Figure 9: Standard curve for HHV6 TaqMan-QPCR for quantification of viral genomes

Quantitative determination of viral genomes by real-time PCR is based on additional use of a
fluorescent probes in a PCR assay. By simultane measurement of a calibration curve based on a
serial dilution of a plasmid standard the number of viral gene copies is detected during
amplification process (Fig. 9).
This extreme sensitive and highly optimized method is unique for estimation of viral loads for DNA
viruses in human tissues and was also applied in the European BICC trial.

9. Autoimmunity Testing
Autoimmune cardiomyopathy is an immune-mediated chronic inflammation of the myocardial
tissue. Induction of autoantibodies could be performed by infection of heart muscle by viruses or by
intramyocardial inflammation. In order to diagnose autoimmunity in patients with cardiac problems
IKDT apply indirect immunofluorescence assays based on BIOCHIP technology of EUROIMMUN
(Germany).
The indirect immunofluorescence test is the analytical method of choice for screening on different
autoantibodies or when it would be too difficult or too complicated to prepare the test antigens
individually for enzyme immunoassays. For the determation of autoantibodies or antibodies against
infectious agents, cells, tissue sections or purified, biochemically characterized substances are used
as antigen substrates.
  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Ultrathin glass slides covered with heart muscle tissue sections of monkey will be incubated with
patient sera. If the sample is positive, specific antibodies in the diluted serum sample attach to the
antigens coupled to a solid phase In a second step, the attached antibodies are stained with
fluorescein-labelled anti-human antibodies and visualized with the fluorescence microscope.
High specificity: positive and negative samples produce a large difference in signal strength. Each
bound antibody shows a typical fluorescence pattern depending on the location of the individual
antigens. Immunofluorescence enables simultaneous detection of antibodies against several
biochemically different antigens on one single biological substrate. Positive samples can be titrated
in steps.




  A                               B                             C
Figure 10: Indirect immunoflurescence detection of autoantibodies against nucleus (ANA) (A),
cross-striated (B) and along-striated muscles (C) on monkey heart tissue by dual green-red
fluorescence microscopy (red: fuscin in cardiomyocytes)

Applying BIOCHIP with monkey heart muscles tissue allows simultan detection of autoantobodies
against nucleus (ANA), mitochondria (AMA), intercalating discs, basal membran, endothelium and
striated muscles (Fig. 10).
Using several BIOCHIPs coated with different substrates side by side on one and the same reaction
field, antibodies against various organs or infectious agents can be investigated simultaneously.
Detailed antibody profiles can thus be established with comparatively little effort, allowing the
reciprocal determination of the results on different substrates.

10. Immunoassays
The immune system protects the body from infection by creating and maintaining barriers that
prevent bacteria and viruses from entering the body. If a pathogen breaches the barriers, and gets
into the body, the innate immune system is equipped with specialized cells that detect, and often
eliminate, the invader before it is able to reproduce, potentially causing serious injury to the host.
The innate immune system protects the host by establishing humoral, chemical and cellular barriers
to infection. Inflammation is produced by chemical factors; including specialized chemical
mediators, called cytokines, and serves as a protective barrier. Cytokine levels in peripheral blood
correspond to systemic situation in patients initiated by various, but often global factors like
infections or inflammatory processes. Cytokines or chemokine are estimated in blood serum for
characterization of present immune response.

  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                                       IKDT
                  Institut Kardiale Diagnostik und Therapie                                                                                                                                                                                     accredited laboratory CAP Nr. 71828-02 since 2003
                                                                                                                                                                                                                                                                                                                 Advancing Excellence



IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


The diagnostic goal of immunology is non-invasive measuring of biomarkers like cytokines and
auto-antibodies characterizing different states of DCM in body fluids (blood), which allow the
monitoring of disease progress or outcome of applied therapy. IKDT offers Multiplex-ELISAs for
measurement of cytokine profiles of DCM patients (Fig. 11).



    1000                                              *

     900
     800                          *                                                     150
                      *
     700                                                                                120

     600
                                                                                         90
     500                                                                        pg/ml
                                                                                          60
     400                                                                                                                                                                                                                                                                                                                                       Hu GM -CSF (34 )
                                                                                                                                                                                                                                                                                                                                             Hu M CP-1(M CA F) (53 )
                                                                                                                                                                                                                                                                                                                                        Hu IL-2 (3 8)
     300                                                                                  30                                                                                                                                                                                                                                         Hu IL-4 (52)

                                                                                                                                                                                                                                                                                                                                  Hu IFN-g (2 1)

     200                                                                                      0                                                                                                                                                                                                                                 Hu IL-12 (p70) (75)

                                                                                                                                                                                                                                                                                                                            Hu IL-1b (3 2)
                                                                      R1
                                                                                                  1216




     100
                                                                                                         1217
                                                                                                                1221
                                                                                                                       1222
                                                                                                                              1224




                                                                                                                                                                                                                                                                                                                       Hu TNF-a (36 )
                                                                                                                                     1225
                                                                                                                                            1226
                                                                                                                                                   1227




                                                                       R2
                                                                                                                                                          1231




                                                                                                                                                                                                                                                                                                                                                      B
                                                                                                                                                                 1232
                                                                                                                                                                        1233                                                                                                                                        Hu IL-10 (56 )
           0                                                                                                                                                                   1234
                                                                                                                                                                                      1237
                                                                                                                                                                                             1238
                                                                            A
                                                                                                                                                                                                    1248
                                                                                                                                                                                                           1249
                                                                                                                                                                                                                                                                                                                Hu IL-8 (54 )




                                                                                                                                                                                                                  1253
                                                                                                                                                                                                                         1254
                                                                                                                                                                                                                                1255
               IL12     TNFa          IFNg




                                                                                                                                                                                                                                       1256
                                                IL8       IL6   IL4




                                                                                                                                                                                                                                              1257
                                                                                                                                                                                                                                                     1262
                                                                                                                                                                                                                                                                                                             Hu IL-6 (19)




                                                                                                                                                                                                                                                            1263
                                                                                                                                                                                                                                                                   1264
                                                                                                                                                                           Patie nte n




                                                                                                                                                                                                                                                                          1265
                                                                                                                                                                                                                                                                                 1266
                                                                                                                                                                                                                                                                                        1267
                                                                                                                                                                                                                                                                                               1268
                                                                                                                                                                                                                                                                                                      1269
                      R1: PVB19+ (n=10)
                      R2: virus negativ (n=9)




Figure 11: Cytokine profiling by single parameters (A) or Multiplex-ELISA of 17 cytokines (B)

The samples for the immunoassay department are mainly patient sera or whole blood. Separation of
sera from EDTA-blood is performed by centrifugation. ELISA are processed immediately after
centrifugation step or aliquots (200 µl) of sera were stored in a freezer (-20°C) until use.
Quantitative detection of cytokines is performed by commercially available immunoassays on full-
automated ELISA system DSX (Fa. ThermoLabsystems, Fig. 11A) or by multiplex ELISA (Fa.
Bio-Rad, Fig. 11B). All standards and controls are included in corresponding kits.
All data for ELISA tests are reported as data files and printed reports.

Cardiomyopathy is a poorly understood disease because it progresses through stages with distinctly
different mechanisms and manifestations finally leading to dilated cardiomyopathy and chronic
heart failure. Most cases of myocarditis result from a viral infection, which may progress to an
autoimmune phase followed by progressive cardiac dilatation. One strategic objective of IKDT is
early detection of auto-antibodies, differential diagnosis and risk assessment of post-viral
autoimmunity in patients suffering from cardiomyopathies.

Detection of auto-antibodies in sera of patients with cardiomyopathies will be performed by use of
commercially available microscopic slides spotted with different tissues which react as antigens.
Processed reaction between sera and specific tissue will be visualized by application of a second
fluorescence labelled antibody. Auto-immunoassays were documented by digital photographs after
fluorescence microscopy.




  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)
                    IKDT
          Institut Kardiale Diagnostik und Therapie
                                                                                                                   Advancing Excellence
                                                                                                accredited laboratory CAP Nr. 71828-02 since 2003


IKDT GmbH, Moltkestrasse 31, D – 12203 Berlin


Summary

IKDT lab offers the most comprehensive approach to analyze myocardial tissue (EMBs) on
morphological abnormalities, viral infections and inflammatory processes as causive reasons for
heart failure problems. The current routine protocol will be expanded continously by new methods
or biomarkers. IKDT is providing submitting institution by diagnostic parameters of examined
patient and also provide you the possiblity to perform clinical trials or be included in running
research project.




 Dirk Lassner, PhD                                                               Prof. Ulrich M. Gross, MD
-Laboratory Director-                                                              -Medical Director-




                                     Prof. Heinz-Peter Schultheiss, MD
                                   -Chairman of Scientific Consulting Board-




  IKDT      Institut Kardiale Diagnostik und Therapie , Moltkestrasse 31 , D-12203 Berlin
          Telefon: +49 (0) 30-8441 5540 | Fax: +49 (0) 30-8441 5555 | Email: info@ikdt.com | Web : www.ikdt.com
              Amtsgericht Berlin-Charlottenburg HRB 83595, UST-Id.Nr. DE 219943700 | Geschäftsführer: Dr. Dirk Laßner
  Ärztlicher Direktor: Prof. Dr. med. U. Gross | Beirat: Dr. T. Kurze (Vorsitz) | Wiss. Beirat: Prof. Dr. med. H.-P. Schultheiss (Vorsitz)

				
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